Paclitaxel induces cognitive impairment via necroptosis, decreased synaptic plasticity and M1 polarisation of microglia

Abstract

Context: Paclitaxel (PTX) leads to chemotherapy brain (chemo-brain) which is characterised by cognitive impairment. It has been reported that necroptosis is associated with cognitive impairment in some neurodegenerative diseases, but it is not clear whether it is related to the development of chemo-brain.

Objective: To investigate the role of necroptosis and related changes in PTX-induced cognitive impairment.

Materials and methods: C57bl/6n mice were randomly divided into five groups: control, vehicle, and different concentrations of PTX (6, 8, 10 mg/kg). Two additional groups received pre-treatment with Gdcl3 or PBS through Intracerebroventricular (ICV) injection before PTX-treatment. Cognitive function, necroptosis, synaptic plasticity and microglia polarisation were analysed.

Results: PTX (10 mg/kg) induced significant cognitive impairment, accompanied by changes in synaptic plasticity, including decreased density of PSD95 (0.65-fold), BDNF (0.44-fold) and dendritic spines (0.57-fold). PTX induced necroptosis of 53.41% (RIP3) and 61.91% (MLKL) in hippocampal neurons, with high expression of RIP3 (1.58-fold) compared with the control group. MLKL (1.87-fold) exhibited the same trend, reaching a peak on the 14th day. The increased expression of iNOS (1.63-fold) and inflammatory factors such as TNF-α (1.85-fold) and IL-β (1.89-fold) compared to the control group suggests that M1 polarisation of microglia is involved in the process of cognitive impairment. Pre-treatment with Gdcl3 effectively reduced the number of microglia (0.50-fold), inhibited the release of TNF-α (0.73-fold) and IL-β (0.56-fold), and improved cognitive impairment.

Conclusion: We established a stable animal model of PTX-induced cognitive impairment and explored the underlying pathophysiological mechanism. These findings can guide the future treatment of chemo-brain.

Keywords: Central neurotoxicity; astrocytes; hippocampus; inflammatory factors; neuron.

Figure 1.

The behavioural test of PTX-treated mice under the different experimental conditions. (A, B, C) Experimental protocol. (D, E) Morris water maze flowchart. (F) The escape latency(s). (G) The number of times the mice passed over the platform. (H) Movement trajectories of mice on 21st day. The results are presented as the mean ± SD, n = 10. In the PTXM group, ^#p < 0.05 vs. vehicle. In the PTXH group, *p < 0.05 vs. vehicle.

Figure 2.

Effect of PTX on synaptic structures and plasticity. (A) Electron microscope images of synapses (scale bar = 500 nm). (B) The width of synaptic cleft. (C) The thickness of post synaptic density. (D) The length of post synaptic density. (E) Golgi staining of the hippocampus of mice. (F) The density of dendritic spines. (G, H) The expression of PSD95 in the hippocampus. (I) The concentration values of BDNF in the hippocampus. The results are presented as the mean ± SD, n = 3. In the PTXH group, *p < 0.05 vs. vehicle, **p < 0.01 vs. vehicle, ***p < 0.001 vs. vehicle.

Figure 3.

The expression of RIP3 and MLKL in the hippocampus of mice treated with PTX. (A) The expression of MLKL at different PTX treatment times. (B) The relative expression of MLKL as shown in A. (C, D, E, F) The expression of RIP3 and MLKL on the 15th day after treatment with PTX. (G, H) Immunofluorescence staining of RIP3 and MLKL (scale bar = 100 μm). (I, J) The deposition of immunocolloidal gold particles observed by high-power electron microscope (scale bar = 500 nm). The results are presented as the mean ± SD, n = 3. In the PTXH group, *p < 0.05 vs. vehicle, **p < 0.01 vs. vehicle, ***p < 0.001 vs. vehicle, ^###p< 0.001 vs. vehicle.

Figure 4.

The expression of RIP3 and MLKL in different types of cells in the hippocampus. (A) Immunofluorescence double-staining of RIP3/MLKL and astrocytes/microglia/neurons (scale bar = 100 μm). (B, C) The proportions of different types of RIP3-positive and MLKL-positive cells. (D) Transmission electron microscopy scans of hippocampal neurons (scale bar = 1 μm or 500 nm). The results are presented as the mean ± SD, n = 3.

Figure 5.

Effect of Gdcl3 on the cognitive impairment induced by PTX. (A) The escape latency(s). (B) The number of times passing the platform. (C) Movement trajectory on the 15th day. (D, E, F, G) The expression of the RIP3 and MLKL proteins. The results are presented as the mean ± SD, n = 10 or 3. In the PTXH + Gdcl3 group, *p < 0.05 vs. PTXH.

Figure 6.

The polarization of microglia involved in PTX-induced cognitive impairment. (A, B, C, D) The expression of iNOS and Arg-1. (E) Polarization of microglia labelled with iNOS/Arg-1 and Iba-1 (scale bar = 50 μm). (F) The proportion of iNOS positive and Arg-1 positive cells to microglia. (G) The number of Iba-1 positive cells. (H, I, J, K) The concentration values of TNF-α, IL-1β, IL-4 and IL-10. The results are presented as the mean ± SD, n = 3 or 5. In the PTXH group, *p < 0.05 vs. vehicle, ***p < 0.001 vs. vehicle. In the PTXH + Gdcl3 group, ^#p < 0.05 vs. PTXH, ^###p < 0.001 vs. PTXH, ^&p < 0.05 vs. vehicle.